Summary

The heart maintains structural and functional integrity during years of
continual contraction, but the extent to which new cell creation participates
in cardiac homeostasis is unclear. Here, we assessed cellular and molecular
mechanisms of cardiac homeostasis in zebrafish, which display indeterminate
growth and possess an unusual capacity to regenerate after acute cardiac
injury. Lowering fish density in the aquarium triggered rapid animal growth
and robust cardiomyocyte proliferation throughout the adult ventricle, greater
than that observed during slow animal growth or size maintenance. Rapid animal
growth also induced strong expression of the embryonic epicardial markers
raldh2 (aldh1a2) and tbx18 in adult epicardial
tissue. Pulse-chase dye labeling experiments revealed that the epicardium
recurrently contributes cells to the ventricular wall, indicating an active
homeostatic process. Inhibition of signaling by Fibroblast growth factors
(Fgfs) decreased this epicardial supplementation of the ventricular wall in
growing zebrafish, and led to spontaneous ventricular scarring in animals
maintaining cardiac size. Our results demonstrate that the adult zebrafish
ventricle grows and is maintained by cardiomyocyte hyperplasia, and that
epicardial cells are added to the ventricle in an Fgf-dependent fashion to
support homeostasis.

INTRODUCTION

Organ homeostasis mechanisms calibrate organ size and function in response
to changing physiological conditions. Perhaps the clearest example of
homeostasis occurs during animal growth, a process accompanied by organ growth
to accommodate increasing demands. For full-sized adult mammals, homeostasis
maintains the status quo, replacing damaged or senescent cells through direct
structural cell proliferation, progenitor cell activity or hypertrophy of
surrounding cells. Some organs are capable of rapid homeostatic adjustments to
tissue loss or gain. For example, the liver will increase mass through
compensatory cell proliferation after partial hepatectomy, or reduce mass
through compensatory cell death after experimentally induced hyperplasia, to
maintain the appropriate size (Conlon and
Raff, 1999; Potter and Xu,
2001; Taub, 2004).
Understanding the cellular and molecular mechanisms of organ homeostasis is a
major research goal, with potential to shed light on therapies for trauma,
degenerative disease and aging.

Although cellular and molecular mechanisms of homeostatic regulation in
most organs are incompletely understood, cardiac homeostasis is particularly
mysterious. For many years, it was believed that the adult mammalian heart is
a post-mitotic organ, and that all postnatal cardiac growth is achieved
through hypertrophy of a fixed number of cardiomyocytes (CMs). Indeed,
analyses of DNA synthesis in the adult murine heart estimated a very low
percentage (a maximum of 0.0005%) of ventricular CMs entering the cell cycle
each day (Soonpaa and Field,
1997). Similar analyses during postnatal rodent growth indicated
that CMs undergo a transition shortly after birth from hyperplastic to
hypertrophic growth, associated with the production of large, multinucleated
CMs through karyokinesis rather than with new, mononucleated CMs through
cytokinesis (Li et al., 1996;
Soonpaa et al., 1996).
Furthermore, there is little evidence of consequential regeneration after
cardiac injuries such as ischemic infarction, and the resulting hypertrophy of
existing muscle that occurs concomitantly with scar formation is detrimental
to cardiac function. The notion that the major structural cells within such a
vigorous and vital organ survive 70 to 100 years in humans without support
from new CMs is contested, especially given recent identification of cell
populations that may possess progenitor activity in the postnatal mammalian
heart (Beltrami et al., 2003;
Laugwitz et al., 2005;
Martin et al., 2004;
Oh et al., 2003). However, a
natural in vivo contribution of these progenitor cells to adult tissue
homeostasis has not been experimentally demonstrated.

The mechanisms by which non-myocardial cardiac cell types are maintained
and replaced are also poorly understood. Interestingly, recent studies have
found that cells of the adult mammalian epicardium, a thin epithelial tissue
surrounding the myocardium proper, can be experimentally stimulated to
differentiate into smooth muscle and endothelial cells in vitro
(Smart et al., 2007;
van Tuyn et al., 2007). In
this way, the in vitro activity of the adult epicardium appears to mimic the
capacity of the embryonic epicardium, a structure that serves as the primary
source of coronary vasculature during heart development
(Reese et al., 2002). Thus, it
is possible that adult epicardial cells also function as a progenitor tissue
to maintain the vasculature or other cardiac cell populations.

Certain non-mammalian vertebrates such as amphibians and fish may have
greater potential for hyperplastic cardiac homeostasis. By contrast with
mammals, many of these species display indeterminate growth, and can rapidly
increase adult mass in response to changes in population density and nutrition
(Jordan, 1905), an increase
that is typically accompanied by organ augmentation. Moreover, some of these
species have demonstrated various degrees of regeneration after mechanical
injury of the cardiac ventricle, a response that may be perceived as a
homeostatic method to restore cardiac mass
(Flink, 2002;
Oberpriller and Oberpriller,
1974; Poss et al.,
2002). In particular, the zebrafish, which displays indeterminate
growth (Tsai et al., 2007),
has a strong cardiac regenerative response, and is amenable to molecular
genetic approaches, represents a unique model system to visualize and dissect
cardiac homeostasis.

Here, we show that adult zebrafish display dramatic, hyperplastic cardiac
growth in response to aquarium conditions that stimulate rapid animal growth,
whereas animals maintaining cardiac size show distinct but rare addition of
new CMs. Additionally, we find that rapid growth conditions induce epicardial
expression of embryonic markers such as raldh2 (also known as
aldh1a2 - Zfin) and tbx18, and that the epicardium regularly
contributes cells to the adult ventricular wall even in the absence of cardiac
growth or myocardial injury. Inhibition of Fgf signaling, a pathway necessary
for normal heart regeneration, disrupts epicardial cell supplementation and
causes spontaneous ventricular scarring in uninjured adult fish. Our study
exposes dynamic myocardial and epicardial mechanisms that mediate cardiac
homeostasis.

MATERIALS AND METHODS

Zebrafish

Zebrafish of outbred Ekkwill or EK/*AB mixed background strains
were raised at a standard density of approx. five fish per liter to 8-10 weeks
of age, at which point fish weighing 70-100 mg were selected for growth
experiments. Fish were placed in conditions that stimulate rapid growth (RG; 3
fish/10 l tank) or returned to conditions that allow normal, slower growth
(SG; 15 fish/3 l tank) for 10-14 days, at which point the animals were weighed
and the hearts extracted. Adult fish, 6-months old, maintaining animal size
(MS) were raised at 3-5 fish/l, the standard in our facility. Ventricular
resection surgeries were performed as previously described
(Poss et al., 2002). All
experiments on animals were performed in accordance with institutional
guidelines and regulations.

To calculate ventricular section surface area, hearts were sectioned
longitudinally, stained with TRITC-phalloidin, and the three largest sections
of the ventricle were imaged and measured for calculation of the area using
Openlab software. The measurements were averaged to give one value for each
heart.

For BrdU incorporation experiments, animals were injected once daily with
2.5 mg/ml BrdU for 3 days prior to collection. Hearts from RG (injected 7, 8
and 9 days after introducing density conditions), SG, and MS animals were
cryosectioned and immunostained for Mef2 and BrdU. Images of the middle of the
lateral ventricle wall (opposite the atrium) from the three largest sections
of each ventricle were selected for analysis. Nuclei labeled with Mef2, a
marker of CMs, BrdU, or both were counted by hand using Adobe Photoshop
images. The region analyzed included the compact muscular wall and a region
approximately six cells thick of trabecular muscle, so that both trabecular
and compact muscle would be included. For these experiments, 150-300
Mef2pos nuclei were counted per section, or 500-900 per animal.

To calculate CM density, Mef2-stained ventricular sections were used to
produce similar images. Then, the area of the region was calculated using
Openlab software, and the number of CM nuclei within the traced region counted
by hand. About 100-300 nuclei were counted per section, or 400-900 per animal.
To assess CM nucleation, ventricles were isolated from cmlc2:nRFP RG,
SG and MS fish (see Results for description of transgenic zebrafish), and
cells were dissociated as described previously
(Warren et al., 2001).
Isolated cells were collected in L-15 medium and live cells imaged to
determine the number of nuclei.

For analysis of RFPcyto cells (see Results for
RFPcyto description), cryosections from cmlc2:nRFP
ventricles were stained with anti-DsRed antibody and the three largest
sections from each heart were used for imaging. For these experiments, images
of the apex were selected for analysis. RFPnuc and
RFPcyto cells were counted by hand from regions including the
ventricular wall and approximately four cell layers of trabecular muscle.
About 150-300 CMs per section were counted, or 600-900 per animal.

For analysis of epicardial-derived cells (EPDCs) within the ventricular
wall, in situ hybridizations for tbx18 were performed on hearts from
wild-type or hsp70:dn-fgfr1 animals under various growth conditions.
Images from the middle of the lateral ventricle wall (opposite the atrium)
were taken from the three largest sections of each heart. For each section,
only tbx18-positive cells clearly separated from the epicardium and
within the compact myocardial wall were counted (see Figs
5 and
6).

RNA isolation and real-time Q-PCR analysis

RNA was collected from 12-15 MS, RG, SG and regenerating [7 days
post-amputation (dpa)] ventricles by extracting the heart in PBS on ice and
mechanically removing the atrium and outflow tracts. Ventricles were
transferred to TRI reagent (Sigma) and the RNA was isolated according to the
manufacturer's instructions, before purification using Qiagen RNAeasy columns.
RNA integrity was assessed by gel electrophoresis, and concentration
determined by spectroscopy. cDNA was made from 200 ng RNA using oligo(dT)
primer and SuperScript III reverse transcriptase (Invitrogen). Quantitative
PCR (Q-PCR) was run using LightCycler FastStart DNA MasterPLUS SYBR
Green I (Roche) on a LightCycler 2.0 machine (software version 4.05x).
Dilutions of cDNA generated from pooled RNA of 20 hours and 56 hours
post-fertilization embryos was used to determine the reaction efficiency (E)
for each primer pair, and ΔCT for each group was calculated
using 7 dpa regenerating hearts as a standard. hand2 expression was
normalized to gapdh for each sample using the following equation:
X=((E)gapdh)-ΔCTgapdh)/((E)hand2)-ΔCThand2).
All samples were run in triplicate, and the averages of two independent
experiments are shown.

Pericardial manipulations

Adult zebrafish ∼6 months of age were placed into 1.5 l tanks (3
fish/l) to maintain a constant density during the experiment. Hearts from
uninjured control animals, as well as manipulated animals, were fixed 3 days
after manipulations. In one group of animals (Open), a single incision was
made in the pericardial sac using iridectomy scissors. In saline-treated
animals, ∼5 μl Hank's buffered saline was injected into the pericardial
cavity from a 30 gauge needle until expansion of the sac was visible. For an
additional group, a needle was similarly inserted into the pericardial sac,
but no fluid was injected (Pierce). Only animals that showed no bleeding after
any of these treatments were used for this study. As a control, iridectomy
scissors were used to make a small incision on the dorsal side of the animal
(Dorsal). For each group 9-11 animals were used in two experimental
trials.

DiI labeling of the adult epicardium

DiI labeling was performed by pipetting ∼0.5 μl of 1 mg/ml Cell
Tracker CM-DiI (Molecular Probes) into the pericardial cavity of∼
6-month-old zebrafish through a small incision in the pericardial sac.
Smaller animals used for RG and SG conditions did not survive this procedure.
Hearts were extracted and fixed at 1 hour, 3 days, 7 days and 14 days
post-labeling. Labeled hearts were sectioned and imaged without coverslips, as
mounting with a coverslip commonly caused diffusion of the dye. At least 15
animals were used for 1 hour, 3 day, and 7 day timepoints in three
experimental trials.

Fgfr inhibition

For RG experiments, heterozygous hsp70:dn-fgfr1 transgenic and
wild-type clutchmates (weighing 70-100 mg each) were selected at 8-10 weeks of
age. Fish received a daily, transient increase in temperature from 26°C to
38°C as described previously (Lee et
al., 2005). Animals remained at 38°C for ∼30 minutes each
day. We found that this temperature increase could not be achieved in 10 l
tanks using our system, so we instead placed one fish each in a 3 l tank, and
performed analyses at 14 days. For adult maintenance experiments, uninjured 4-
to 6-month-old hsp70:dn-fgfr1 and wild-type adult clutchmates
received a daily temperature increase for 60-70 days.

Experimental control of animal and cardiac growth in adult
zebrafish. (A) Representative images of hearts collected from
animals maintained for 14 days in conditions for rapid growth (RG), slow
growth (SG) or to maintain size (MS). Hearts in images may be missing some
atrial tissue lost during tissue collection. a, atrium; b, bulbous arteriosus
v, ventricle. (B) Quantification of animal mass of RG, SG and MS fish.
RG fish had a much higher increase in body mass from day 0 than their SG
clutchmates at day 14, whereas MS fish maintained mass
(*P<0.001, t-test, significantly different
from day 0). The numbers of animals per group are shown in parentheses.
(C) Quantification of ventricular size in RG, SG and MS fish.
Ventricles in the RG group showed much greater size increases than those of SG
clutchmates at day 14, while ventricular size remained stable in MS fish
(*P<0.005, t-test, significantly different
from day 0).

RESULTS

Experimental control of adult cardiac homeostasis

We predicted that the vigor of cardiac homeostasis would be proportional to
the rate of adult animal growth. Therefore, we developed three conditions
based on aquarium density that we expected to stimulate rapid growth (RG),
slow growth (SG) or size maintenance (MS). We found that when small adult fish
(8- to 10-weeks old, 70-100 mg) were removed from standard aquarium density
conditions (SG; 5 fish/l) and placed in low-density conditions (RG; 0.3
fish/l), body mass tripled after only 14 days (a ∼208% increase from day
0). Clutchmates maintained at SG displayed only a ∼13% increase in mass
over this period. Fully mature, 6-month-old fish, considerably larger at the
onset of experiments than RG and SG fish, maintained mass for at least 28 days
when kept at standard aquarium density conditions
(Fig. 1B). In general we found
that starting size was a greater determinant of animal growth rate than age,
consistent with other published studies
(Tsai et al., 2007).

In these experiments, the large increases in body mass seen in RG fish were
accompanied by dramatic increases in the size of both heart chambers
(Fig. 1A). To quantify changes
in ventricular size, we measured the surface area of longitudinal ventricular
sections using digital imaging software. These analyses revealed that
ventricular size more than tripled after only 14 days in RG conditions (a∼
221% increase from day 0), a rate of cardiac growth approximately
equivalent to that of animal growth under these conditions. Clutchmates in SG
conditions exhibited a more modest increase in ventricular size over this
period (∼48%). Because this increase was greater than the increase in
animal size during this period, it is possible that the rate of cardiac growth
may slightly diverge from the rate of animal growth in SG conditions. MS
animals experienced no significant change in ventricular size over 14 days
(Fig. 1A,C). Thus, by varying
aquarium density and starting size of the animal, we closely controlled the
rate of cardiac homeostasis. In particular, low aquarium density conditions
induced remarkably rapid cardiogenesis in adult zebrafish.

New cardiomyocyte creation accompanies cardiac growth and
maintenance

To determine whether density-dependent control of ventricular size resulted
from hyperplasia or hypertrophy, we administered three daily injections of
BrdU prior to harvesting tissue after 9 days in RG, SG or MS conditions. CMs
were specifically assayed for BrdU incorporation by costaining with an
antibody against Mef2, a transcription factor that regulates myocardial
differentiation (Molkentin and Markham,
1993). We observed a high rate of BrdU incorporation in CMs within
both trabecular and compact muscle compartments of RG fish, with
10.1±1.8% of CMs labeled by BrdU (mean ± s.e.m.;
Fig. 2A,B). This labeling index
was almost eight times that of SG animals (1.3±0.3%), and 25 times that
of MS animals sustaining constant body mass and ventricular size
(0.4±0.1%; Fig. 2A,B).
We obtained similar results when identifying proliferating cells with an
antibody against PCNA (see Fig. S1 in the supplementary material). We suspect
that rare CM proliferation in MS animals serves to replace dying cells, as we
observed occasional CM apoptosis events by TUNEL staining of ventricular
sections (see Fig. S2 in the supplementary material).

To assess possible contributions of hypertrophy to growth, we measured the
density of cells positive for the nuclear marker Mef2 in ventricular sections.
There were no differences in CM nuclear density per myocardial area in RG, SG
and MS fish, indicating a lack of CM hypertrophy
(Fig. 2C). In addition, we
dissociated ventricles and assessed the extent of binucleation, a consequence
of karyokinesis without cytokinesis that is common in adult mammalian CMs. We
found that the vast majority of ventricular CMs in rapidly growing fish were
mononucleate (95.6%), as were CMs from fish kept at SG (97.9%) and MS
conditions (95.1%), indicating derivation through cytokinesis
(Fig. 2D,E). Thus, homeostatic
cardiac growth and maintenance are primarily the result of bona fide CM
hyperplasia, with the vigor of this hyperplasia dependent on the rate of
animal growth.

Evidence that myocardial homeostasis is aided by progenitor
cells

Homeostatic CM generation must occur through the division of mature CMs, or
the maturation of myocardial progenitor cells, or both. During regeneration,
cells expressing markers of embryonic cardiac progenitor cells such as
hand2 (Yelon et al.,
2000) are established at the apical edge of the wounded muscle
within 3-4 days post injury, and are maintained at that edge as regeneration
progresses (Lepilina et al.,
2006). The origin of these cells is unknown; they might exist as
resident non-myocardial cells activated upon injury, or be created through
reduction in contractile function of existing CMs, known as
de-differentiation. Strong, discrete hand2 in situ hybridization
signals analogous to that seen during myocardial regeneration were common in
RG animals particularly within the compact myocardium, sparingly present in SG
animals, and largely absent in MS adults
(Fig. 3A). Real-time
quantitative PCR experiments indicated that hand2 expression in RG
ventricles was 1.4 fold that of SG clutchmates, and 2.5 fold that of MS
ventricles (Fig. 3C).

Cardiac homeostasis involves cardiomyocyte hyperplasia. (A)
Ventricles from SG, RG and MS animals stained for Mef2 expression to identify
CMs (red), and for BrdU incorporation (green). CM BrdU incorporation was much
greater in RG ventricles (arrowheads in insets). Nuclei are labeled with DAPI
(blue). (B) Quantification of CM BrdU incorporation in SG, RG and MS
groups, plotted as the average BrdU incorporation index per animal
(*P<0.001, t-test, significantly different
from SG; **P<0.05, t-test, significantly
different from SG and RG). Numbers above the error bars indicate BrdU-positive
CMs over the total CMs counted from all animals combined. Numbers below group
abbreviations indicate the number of animals/ventricles per group. (C)
CM hypertrophy was assessed by measuring the number of Mef2-positive nuclei
per area of myocardium, as described in Materials and methods. No significant
differences in CM density were observed between RG ventricles (6540±505
CMs/mm2) and SG (5842±326 CMs/mm2) or MS
ventricles (5167±422 CMs/mm2). Eight animals were used per
group. (D) Quantification of nuclei in CMs dissociated from pooled
cmlc2:nRFP ventricles from different homeostatic conditions,
indicating that 97.9% of CMs are mononucleate in SG ventricles, 95.6% in RG
ventricles and 95.1% in MS ventricles. (E) Image of dissociated
ventricular CMs (cells with white nuclei) from cmlc2:nRFP animals.
Binucleated myocytes (arrowhead) are rare in adult zebrafish under RG, SG, or
MS conditions. Thus, CM hypertrophy and binucleation play little or no role in
homeostatic cardiac growth. Scale bar in A: 100 μm.

hand2 is expressed in embryonic progenitors but is not a
definitive marker of undifferentiated cells. To test the idea that progenitor
cell populations give rise to new CMs during homeostasis, we assessed CM
maturation in the cmlc2:nRFP transgenic line, a strain that reports a
slow-folding, nuclear-localized DsRed2 protein under control of the
cardiac myosin light chain 2 (myl7 - Zebrafish Information
Network) promoter (Mably et al.,
2003). Mature CMs in these transgenic fish show nuclear-localized
DsRed fluorescence. However, an anti-DsRed antibody detects unfolded,
cytosolic DsRed fluorescence in the new CMs of the 24-hour post-fertilization
embryo and the regenerating edge of the injured adult ventricle, apparently
before nuclear localization occurs and natural fluorescence is manifest. Thus,
this marker (RFPcyto) appears to serve as an indicator of
developmental transition from undifferentiated progenitor cells to
differentiated CMs (Lepilina et al.,
2006). We found that 10.7±2.1% of CMs throughout the
ventricle of RG animals expressed the RFPcyto marker, a percentage
over three times that of SG clutchmates (3.1±0.3%) and 36 times that of
MS fish (0.3±0.1%; Fig.
3B,D). In ventricles of cmlc2:nRFP RG animals
that had been injected with BrdU, both RFPcyto and
RFPnuc CMs showed BrdU-labeled nuclei, suggesting that both the
most recently maturing CMs and earlier-maturing CMs undergo proliferation
(data not shown). In total, these results suggest that myocardial progenitor
cell populations contribute at least in part to the vigorous generation of new
CMs stimulated by rapid animal growth, and in rare events during homeostatic
maintenance of heart size.

Homeostatic developmental activation of the adult epicardium

During zebrafish heart regeneration, local injury strongly activates
expression of the embryonic epicardial markers raldh2 and
tbx18 throughout the entire ventricular and atrial epicardium. This
activation is accompanied by epicardial cell proliferation, expanding the
epithelial cover of the ventricle to eventually cover the injury
(Lepilina et al., 2006).
raldh2 and tbx18 were expressed at low levels in a small
number of epicardial cells in both SG and MS ventricles, indicating moderate
activation. By contrast, these markers were robustly induced in contiguous
stretches of RG atrial and ventricular epicardium
(Fig. 4A,
Fig. 5B). In addition,
endocardial cells surrounding cardiac myofibers near the injury site have been
shown to induce raldh2 during regeneration (R.J.M. and K.D.P.,
unpublished) (Lepilina et al.,
2006). We found that RG animals, but not SG or MS animals,
displayed strong raldh2 expression in endocardial cells throughout
the ventricle (arrows in Fig.
4A; A.A.W., J.E.H. and K.D.P., unpublished). Thus, embryonic gene
expression within both the epicardium and endocardium is activated by rapid
animal growth.

Cardiogenic markers are induced during cardiac homeostasis.
(A) hand2 in situ hybridization of ventricular sections.
hand2 is weakly expressed in MS hearts, mildly expressed in SG hearts
and more strongly expressed in RG hearts similar to regenerating hearts 7 days
post-resection (arrowheads show foci of strong expression). (B)
Sections from SG, RG and MS ventricles of cmlc2:nRFP zebrafish,
stained for DsRed immunoreactivity. RG ventricles show many more
RFPcyto cells, a marker that suggests recent CM differentiation
events (arrowheads in insets). (C) Real-time Q-PCR analysis of
hand2 expression from whole ventricles of SG, RG and MS hearts,
relative to expression in ventricles 7 days after apical resection.
hand2 expression is similar in ventricles from RG fish to that in
regenerating ventricles (1.0, represented by dashed line) and greater than in
SG and MS ventricles. (D) Quantification of RFPcyto cells in
SG, RG and MS groups. The average RFPcyto index per animal is
plotted. Numbers above the error bars indicate RFPcyto CMs over the
total CMs counted from all animals combined. Numbers below group abbreviations
indicate the number of animals/ventricles per group
(*P<0.01, t-test, significantly different from
SG; **P<0.005, t-test, significantly different
from SG and RG). Scale bars: 100 μm.

We suspected that, because of its location and exquisite sensitivity to
growth and injury, the epicardium may act as a sensor to identify changes in
animal growth through sensation of pressure changes in the extra-cardiac
space. The pericardial sac is filled with a plasma ultrafiltrate that is
believed to have a restrictive function during diastole. Therefore, opening
the cavity might relieve pressure, increasing cardiac stretch. Conversely,
saline injection is expected to increase pericardial pressure and limit the
ability of the heart to expand (Farrell et
al., 1988; Shabetai et al.,
1985; Spodick,
1997). To test whether these procedures affect epicardial gene
expression, we examined raldh2 expression in several groups of
experimentally manipulated 6-month-old MS animals. We found that ventricular
raldh2 expression was induced to roughly the same level as in RG
animals (but at levels lower than after partial ventricular resection) in
experiments in which (1) the pericardial sac was surgically opened, or (2)
saline was carefully injected into the pericardial cavity
(Fig. 4B). Different
manipulations that resulted in no effect on epicardial raldh2
expression included: (1) piercing the pericardial sac without saline
injection, (2) lesions to the dorsal region of the animal and (3) fin
amputation. These results show that the epicardium is a dynamic tissue
responsive to changes in the milieu surrounding the heart, and suggest that
the effects of rapid animal growth on epicardial gene expression are due in
part to an ability of the epicardial tissue to sense changes in resistance
within the extra-cardiac space.

Epicardial-derived cells recurrently supplement the ventricular
wall

During embryonic heart development, a subset of epicardial cells undergoes
epithelial-mesenchymal transition (EMT) and migrates into the underlying
subepicardial space and myocardial wall as epicardial-derived cells (EPDCs).
There, EPDCs contribute fibroblasts as well as smooth muscle and/or
endothelial cells for building coronary vasculature
(Olivey et al., 2004;
Reese et al., 2002). To
determine whether the adult zebrafish epicardium actively transfers cells
subepicardially to the ventricular wall, we performed a pulse-chase
experiment. We labeled the epicardium by filling the pericardial sac of MS
zebrafish with the fluorescent lipophilic dye DiI, and then harvested hearts
at different timepoints post-injection. Dye was limited to the epicardium at 1
hour post-injection; by contrast, we noticed a small number of labeled cells
separated from the epicardium by 3 days post-injection. Occasionally, these
labeled cells had a tubular morphology reminiscent of vascular tissue (inset,
Fig. 5A). At 7 and 14 days
post-injection, DiI was concentrated in cells that were embedded within the
otherwise unlabeled ventricular wall inward from the now faintly labeled
epicardium (Fig. 5A).
Interestingly, labeled cells occasionally appeared to constitute a distinct
layer of 15-20 μm at the junction of compact and trabecular myocardium.
These surprising data indicate that the adult epicardium is a dynamic tissue
that regularly contributes EPDCs to the ventricular wall, presumably through
EMT.

Developmental activation of the epicardium in response to growth or
manipulation of the extracardiac environment. (A) Ventricles from
SG, RG, or MS animals stained for raldh2 expression. raldh2
is weakly expressed in rare cells of SG and MS epicardia (arrowheads), but
strongly expressed in epicardial cells of RG hearts, similar to the expression
seen at the wound at 7 days post-amputation (right). Ventricular endocardial
cells surrounding inner trabecular myofibers also induced raldh2
during rapid growth, similar to induction during regeneration (arrows in RG
and Regeneration). (B) Assessment of raldh2 expression in
ventricular epicardium 3 days after different manipulations in 6-month-old MS
animals (lateral ventricular wall is shown). Surgically opening the
pericardial sac (Open) or injecting saline into the pericardial sac (Saline)
stimulated raldh2 expression (arrowheads), whereas injuries to the
dorsal side of the animal (Dorsal), or piercing the pericardial sac with an
empty needle (Pierce) did not. Scale bars: 100 μm.

Applying labeled dye to the pericardium elevated epicardial expression of
raldh2 similar to saline injection (data not shown), implying that
this treatment induces strong developmental activation of the epicardium and
likely affected its contributions to the ventricular wall. To evaluate EPDC
activity in unmanipulated animals, we utilized the embryonic epicardial marker
tbx18. During regeneration, cells positive for the embryonic
epicardial marker tbx18 emerge not only in the epicardium, but in
subepicardial tissue within the wound and regenerating muscle
(Lepilina et al., 2006).
Interestingly, in uninjured ventricles, tbx18-expressing cells were
also present both in the epicardium and several cell layers deep into the
compact myocardium. Many tbx18-positive cells were located in what
appeared as a distinct layer 15-20 μm subepicardially, a location similar
to the EPDCs revealed by our pulse-chase experiments
(Fig. 5B). For these reasons,
we inferred that tbx18 is a robust marker for emergent EPDCs in the
uninjured ventricular wall. Quantification of these internalized
tbx18-positive EPDCs revealed that the density of EPDCs in RG animals
was almost six times as high as in SG animals (27.4±1.6 vs
4.9±1.0 tbx18-positive cells/mm ventricular wall). Somewhat
unexpectedly, MS animals had more than twice as many tbx18-positive
EPDCs as SG animals (11.9±1.8; Fig.
5C). This may be a consequence of age differences between these
groups, and/or the possibility that tbx18 is a cumulative marker of
EPDCs that have emerged in the ventricular wall over weeks to months. The
latter idea is supported by the observation that regenerated muscle possesses
cells robustly expressing tbx18 for 30 days or more after injury
(Lepilina et al., 2006). Given
the role of epicardial tissue during embryonic heart growth and the appearance
of tbx18-positive cells coincident with new vascular tissue during
regeneration, we postulate that tbx18-positive EPDCs contribute to
neovascularization of emergent myocardial tissue and/or vascular maintenance
in uninjured animals. Indeed, we could observe in some cases cells positive
for tbx18 that had the appearance of vascular tissue
(Fig. 5B, red arrowhead and
inset), similar to DiI-labeled cells in pulse-chase experiments. Thus, the
adult epicardium supports cardiac homeostasis by regular contribution of new
EPDCs to the ventricular wall.

Contribution of epicardial-derived cells (EPDCs) to the
ventricular wall. (A) DiI was carefully injected into the
pericardial sac to label the epicardium, and hearts were collected at 1 hour,
3 days and 7 days post-injection. Dye is restricted to epicardial cells at 1
hour, but at 3 days EPDCs are occasionally seen in the compact layer
(arrowheads). The red arrow indicates labeled tissue with the appearance of
vascular tissue (enlarged in the inset). By 7 days post-injection,
DiI-containing EPDCs are observed deep in the ventricular wall, with less
label within the epicardial layer. Many of these cells appear to contribute to
an inner layer of labeled cells ∼15-20 μm into the wall. (B)
tbx18 is expressed in many more epicardial cells and EPDCs
(arrowheads) in RG animals than in other groups. (Inset) Enlarged image of
area indicated by the red arrowhead: some tbx18-positive EPDCs within
the ventricle showed a morphology characteristic of vascular cells. The red
dotted line in injured animals indicates the border between myocardium and
clot material. (C) Quantification of tbx18-positive EPDCs from
SG, RG and MS ventricles. (*P<0.001, t-test,
significantly different from SG; **P<0.01,
t-test, significantly different from RG and SG). Scale bars: 100μ
m.

Cardiac homeostasis requires Fibroblast growth factor signaling

Recent studies have revealed roles for Fgfs in epicardial EMT during both
embryonic heart development and adult heart regeneration. EMT of cultured
embryonic epicardial cells can be stimulated by Fgfs in vitro, and this
pathway is necessary for normal coronary vascular development in mice
(Lavine et al., 2006;
Morabito et al., 2001). During
zebrafish heart regeneration, the injured zebrafish myocardium increases
expression of fgf17b, while the receptors fgfr2 and
fgfr4 are expressed in EPDCs. Furthermore, abrogation of Fgf
signaling disrupts EPDC supplementation of the wound and new muscle during
regeneration, inhibiting neovascularization and myocardial renewal
(Lepilina et al., 2006). We
found that fgf17b, fgfr2 and fgfr4 were all present in SG
and RG ventricles, although no differences in expression between the two
groups were detectable by in situ hybridization (data not shown). Based on
these findings, we suspected that Fgf signaling may also be important for EPDC
supplementation of the ventricle during cardiac homeostasis.

To test this idea, we placed zebrafish transgenic for a heat-inducible
dominant-negative Fgf receptor (hsp70:dn-fgfr1) and their wild-type
clutchmates in RG conditions for 14 days, and applied a daily heat-shock
(Lee et al., 2005). Transgenic
animals appeared healthy during the experiment, although they grew less than
wild-type animals under RG conditions (see Fig. S3 in the supplementary
material). Although tbx18 expression was comparable between
transgenics and wild types in the epicardium itself, the density of
tbx18-positive EPDCs within the ventricular walls of
hsp70:dn-fgfr1 animals was less than half that in wild-type animals
(Fig. 6A,B; 24.6±2.1
EPDCs/mm in wild type, versus 11.5±2.1, in hsp70:dn-fgfr1).
Fgfr inhibition also resulted in a slight reduction in ventricular growth
under RG conditions, with transgenic animals exhibiting ∼31% less of a
growth increase than wild-type clutchmates
(Fig. 6C). Thus, normal
homeostatic supplementation of the ventricular wall by EPDCs requires Fgf
signaling. We suspect that the partial phenotype is due to the continued
presence of other factors that act on EPDCs, incomplete inhibition of Fgf
signaling, and/or the possibility mentioned above that tbx18
expression reflects cumulative events of EPDC contribution.

As pulse-chase and tbx18 expression experiments indicated ongoing
EPDC contribution even in the absence of significant cardiac growth, we tested
the requirements for Fgf signaling in mature adult zebrafish. We delivered a
long-term block of Fgf signaling by daily heat-shocks to
hsp70:dn-fgfr1 transgenics for 60-70 days, with identical heat-shocks
given to wild-type clutchmates. We found that ∼15% of
hsp70:dn-fgfr1 fish developed extensive collagen deposition within
the ventricular wall after this period, indicating the presence of scar tissue
(Fig. 6D; n=39 fish).
No wild-type animals displayed scarring (n=44 fish; Fisher-Irwin
test, P<0.01). We suspect that these fibrotic events resulted from
reduced efficiency of incremental EPDC supplementation over the 2-month
period, although marker examination revealed no obvious defects (data not
shown). These results demonstrate that Fgf signaling is essential for normal
homeostatic growth and maintenance of cardiac tissue in the adult
zebrafish.

DISCUSSION

Hyperplastic myocardial and epicardial homeostasis in the adult
zebrafish ventricle

We have discovered that new myocardial and epicardial-derived cells are
created during homeostatic responses of the adult zebrafish heart, helping to
couple cardiac and animal growth rate and maintain cardiac integrity. One
molecular signaling pathway essential for these responses is that mediated by
Fgfs, which ensures normal supplementation of the ventricular wall with EPDCs
and helps to prevent scarring.

Fgf signaling is required for cardiac homeostasis.
(A,B) tbx18 expression in ventricles of wild-type and
hsp70:dn-fgfr1 transgenic zebrafish after 14 days RG conditions, with
a single daily heat-shock. Although epicardial expression is comparable,
significantly fewer EPDCs (arrowheads) are observed within
hsp70:dn-fgfr1 ventricular walls (*P<0.001,
t-test, n=10). (C) Fgfr inhibition attenuates
ventricular growth of RG fish (*P<0.001,
t-test, significantly different from day 0;
**P<0.01, t-test, significantly different from
day 0 and from day 14 wild type, n=9). Day 0 measurements are the
results of pooled transgenics and wild types. (D) Acid-fuschin-Orange G
staining (AFOG) on ventricles of wild-type and hsp70:dn-fgfr1 animals
after 60-70 days of daily heat-shocks. Scars (brackets) were observed in∼
15% of uninjured transgenic fish (n=39), but never observed in
wild-type clutchmates given the same daily heat-shock protocol (n=44)
(P<0.01, Fisher-Irwin exact test). Scale bars: 100 μm.

In contrast to what is known about postnatal ventricular growth in mammals,
we have found that cardiac growth in zebrafish is a result of CM
proliferation, not the hypertrophy of existing cells. Ventricular expression
of the embryonic progenitor marker hand2, as well as a transitional
marker for newly generated cardiomyocytes in cmlc2:nRFP transgenic
fish, showed positive correlations with cardiac growth and CM proliferation.
Thus, these results suggest that undifferentiated progenitor cells contribute
to CM hyperplasia. Future experiments to address the extent to which this
occurs must involve genetic fate-mapping strategies
(Cai et al., 2003;
Dor et al., 2004;
Hsieh et al., 2007;
Meilhac et al., 2003).
Moreover, it will be critical to define homeostatic signals that trigger
cardiomyogenesis and how these signals are distributed and calibrated in the
adult animal.

As a thin epithelium covering the entire surface of the heart, the
epicardium has an ideal architecture for relaying such homeostatic signals.
Numerous studies support this idea, because: (1) the epicardium serves as a
source of mitogens in the embryonic heart
(Chen et al., 2002;
Merki et al., 2005;
Reese et al., 2002), (2)
organ-wide developmental gene expression is activated within the adult
zebrafish epicardium within hours of cardiac injury
(Lepilina et al., 2006), and
(3) during regenerative cardiogenesis as well as homeostatic cardiac growth,
developmentally active epicardium is present at sites of cardiogenesis
(Lepilina et al., 2006).
Indeed, consistent with this idea are the findings here that manipulations of
the pericardial environment that mimic growth-induced changes in pericardial
space cause epicardial responses similar to those elicited by homeostatic
growth. Because epicardial retinoic acid (RA) synthesis is regulated in each
of these scenarios and is known to influence CM proliferation in embryos
(Chen et al., 2002;
Stuckmann et al., 2003), this
molecule is a strong candidate for homeostatic regulation of cardiogenesis. We
have found that the endocardium also increases RA synthesis during adult
growth and regeneration. It is possible that endocardial cell activity
regulates growth and/or remodeling of the inner trabecular muscle, which would
appear to have reduced access to epicardial signaling molecules.

In addition to serving as a source of RA, we found that the epicardium also
regularly contributes cells to the compact myocardial wall of the ventricle.
Thus, the epicardium is by no means a static tissue in adult animals, but
rather one that actively sustains the ventricle. This conclusion is bolstered
by our finding that sustained inhibition of Fgf signaling, which disrupts EPDC
supplementation in rapidly growing fish, led to spontaneous scar formation in
animals maintaining their animal and organ size. Further exploration of the
fate and function of these adult EPDCs, using tissue-specific fate-mapping and
ectopic expression tools, will illuminate the roles of the epicardium in
cardiac homeostasis. From what is known of embryonic heart development, it is
likely that the adult zebrafish myocardial wall requires new EPDCs to
replenish vasculature, or to neovascularize newly created muscle, or both. It
will also be fascinating to learn to what extent the epicardium participates
in homeostasis of the adult mammalian ventricular wall.

Cardiac growth, maintenance and regeneration

Our study exposes important similarities between cardiac homeostasis and
cardiac regeneration. In each case, new myocardial and epicardial-derived
tissues are generated in amounts appropriate to animal size, involving
expression of a suite of cardiogenic developmental markers. Whereas
regeneration is characterized by an intense focus of cardiogenesis at the
injury site, homeostasis involves organ-wide cell addition in doses dependent
on the vigor of animal growth. Thus, the adult zebrafish not only has the
ability to generate new cardiac tissues, but can also control the intensity
and the spatial distribution of these cardiogenic events to select replacement
of a portion of tissue, rapid chamber-wide growth, or occasional exchange of a
few cells (Fig. 7).

Model for cardiac homeostasis in zebrafish. Regeneration,
maintenance and growth share cardiogenic signaling pathways (black arrows
above ventricles) to generate new CMs and EPDCs in adult zebrafish.
Regeneration involves developmental activation of the epicardium (black dots)
and local recruitment of EPDCs to a focus of new CM production (pink
explosions) at the wound (red dotted line). For simplicity, a cartoon of the
ventricle at 7 dpa is shown, after the epicardial response has localized to
the injury site. In the uninjured MS ventricle (maintenance), CM generation,
epicardial activation and EPDC recruitment are diffuse and rare to counter
occasional cell loss events. During rapid animal growth (growth), robust
chamber-wide increases in CM generation, epicardial activation and EPDC
mobilization are stimulated. Thus, similar or identical cardiogenic pathways
are regulated with different intensities and localization to facilitate
cardiac regeneration, maintenance or growth in adult zebrafish.

This relationship between cardiac regeneration, homeostatic growth and
homeostatic tissue maintenance is likely to be important for explaining why
zebrafish regenerate injured cardiac muscle. Both regeneration and homeostasis
stand to benefit from sophisticated mechanisms to activate and deploy
epicardial and myocardial cells for cardiogenesis. In some teleosts, adult
growth rate is controlled by environmental and/or social factors and can
influence mating preference (Borowsky,
1973; Hofmann et al.,
1999). Thus, one reason why cardiac regeneration persists in
zebrafish, and probably other teleosts, may be the accessibility of injured
tissue to cellular and molecular machinery that is normally employed for rapid
indeterminate growth. This notion makes sense in light of extremely weak
hyperplasia in the injured or uninjured hearts of adult mammals, species with
determinate growth. Continued study of cardiac regeneration and homeostasis in
zebrafish, including comparisons with mammalian cardiac dynamics, will expose
critical mechanisms by which cardiac tissues are rejuvenated.

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